Colon and rectal diseases constitute an extensive group of pathological conditions affecting human large bowel. The importance of early detection and treatment of colorectal diseases is generally recognised, but the range of existing non-invasive screening and diagnostic tests remains strictly limited. Colorectal cancer (CRC) and inflammatory bowel disease (IBD) constitute two major groups of large bowel disorders deserving special attention.
Colorectal cancer (CRC) is one of the most prevalent malignancies with over 1,300,000 new cases detected annually worldwide. The disease mostly affects people over the age of 50 in developed Western countries (Ferlay et al., 2010). Although CRC is the second leading cause of oncological mortality, the disease is curable if detected early. Unfortunately it is often diagnosed late (Richards, 2009) because in many cases clinical manifestations do not appear until advanced CRC stages. For this reason introduction of active screening of asymptomatic people of older (over 50) age groups for CRC presence is regarded as the only possible way to radically improve early cancer detection and patient survival rates.
An ideal cancer screening test should be efficient for disease detection (highly sensitive and specific), non-invasive, safe, well-tolerated, simple, inexpensive, easily repeatable and preferably self-applicable.
Flexible colonoscopy is currently regarded as the most sensitive and specific diagnostic procedure for colorectal tumour detection, but it is an invasive investigation requiring bowel preparation and occasionally causing serious complications (Ransohoff, 2009). It is also important that colonoscopy is an expensive procedure, which should be performed only by highly qualified and specially trained medical professionals. Although flexible colonoscopy is correctly defined as the final common pathway of every colorectal screening program (Lieberman, 2009), the idea of introducing it as the only valid approach to CRC screening advocated by some US experts does not constitute a viable option for most countries (Hoff et al., 2010).
The necessity of a simple, non-invasive and inexpensive test, which could be used as the “first line” of CRC screening is a long-standing problem in clinical medicine. For the last few decades this place has been occupied by Faecal Occult Blood Test (FOBT), first proposed for CRC detection by Greegor (Greegor, 1969). The test is based on the assumption that these tumours often bleed; hence blood presence in stool may indicate bleeding from a colorectal tumour.
U.S. Pat. Nos. 3,252,762; 3,996,006; 4,092,120; 4,199,550; 4,333,734; 4,562,043; 4,939,097; 5,391,498; 5,563,071 describe different versions of the conventional guaiac-based test detecting the presence of haemoglobin in faeces, but these tests may give false-positive results due to the presence of residual haemoglobin in food remnants. A more precise modern version of the test, which is human haemoglobin-specific through immunochemical detection of the protein (globin) component of the human haemoglobin is called Immunochemical Faecal Occult Blood Test (iFOBT) or Faecal Immunochemical Test (FIT). This type of test is exemplified by the methods described in U.S. Pat. Nos. 4,427,769; 5,198,365; 7,288,413.
Although FOBT and iFOBT have some attractive characteristics, being non-invasive, simple, cheap and easily repeatable, application of these tests frequently produces false-positive and especially false-negative results (Allison et al, 1996; 2007; Collins et al, 2005; Burch et al, 2007; Duffy et al, 2011) reflecting the fact that the presence or absence of blood in stool is often unrelated to the presence of CRC (Itzkowitz, 2009). Indeed, in many cases colorectal tumours do not bleed, whereas bleeding from non-neoplastic conditions such as haemorrhoids is very common. Another minor drawback of this test is related to the necessity of temporarily preserving excreted stool for collecting samples for analysis.
The idea of using colonic epithelial cells (colonocytes), which are exfoliated from colonic mucosa and the surface of colorectal tumours, has attracted clinical investigators since the early report of Bader and Papanicolau on cytological differences between material obtained using rectal washings from CRC patients and normal individuals (Bader et al, 1952). Consideration of a possible clinical application of this approach is reflected in U.S. Pat. No. 3,735,751. This patent describes an instrument for rectosigmoid lavage and liquid material collection for cytological investigation. Nevertheless, the lavage-based method of material collection was invasive and unreliable and has never been introduced into clinical practice.
Renewed interest in using exfoliated colonocytes was provoked by studies of P. P. Nair and his group, who claimed that it was possible to isolate “thousands of viable exfoliated colonocytes” from dispersed human stool samples by centrifugation in a density gradient (lyengar et al., 1991). This assertion constituted the basis of a family of patents obtained by P. P. Nair including U.S. Pat. Nos. 6,355,193; 6,534,280; 6,630,314; 6,645,729; 6,645,730; 6,881,574. Nevertheless, no cytological evidence of colonocyte presence in preparations made according to the patents has been provided by the authors. For this reason the correctness of the key claim regarding viable colonocyte isolation from faeces in significant numbers has been strongly disputed (Loktionov et al, 1998; Loktionov, 2007). Eventually the approach has never been used for clinical purposes.
U.S. Pat. No. 5,981,651 suggested application of epithelium-targeting immunomagnetic beads for recovering epithelial cells from small stool samples, but the method appeared to be complex. Moreover, the recovered cells could only be subjected to qualitative molecular analysis, which still remains to be established as a credible approach to solving clinical problems (see below).
Cell exfoliation is an important mechanism of epithelial tissue renewal, which is relatively inactive in the colorectal epithelium in normal physiological conditions and becomes dramatically activated when neoplastic growth occurs (Loktionov, 2007). Cells shed from normal or neoplastic epithelium are first incorporated into a well oxygenated layer of mucus overlaying the mucosal surface (Matsuo et al., 1997). This mucocellular layer, which provides protection to the exfoliated cells, gradually moves distally alongside faecal flow (Loktionov, 2007; Loktionov et al., 2009). It is inevitable that elements of the mucocellular layer are always excreted with stool during defaecation, but their presence should be mostly confined to stool surface. The validity of this assumption was successfully proven both in experimental (Loktionov et al., 1995) and human (Loktionov et al., 1998) studies and constituted the core of U.S. Pat. No. 6,187,546. The method of exfoliated cell isolation described in that patent provided convincing evidence of the presence of well-preserved easily morphologically identifiable colonocytes in the collected material (Bandaletova et al., 2002). The suggested clinical application of the approach was related to the detection of higher total DNA amount in stool samples from CRC patients compared to cancer-free individuals. Loktionov et al. confirmed that the use of stool surface washes provides much better discrimination between these groups (Loktionov et al., 1998), but the method described in U.S. Pat. No. 6,187,546 required collection and treatment of whole stool samples. This requirement made its wide application for clinical purposes impossible.
Other techniques employing stool DNA for detecting molecular biomarkers of CRC are mostly based upon gene mutation detection initiated following work on mutant k-ras detection in stool samples of some CRC patients. This family of methods involves PCR amplification of selected gene regions of human DNA isolated from stool. Various approaches of this type are described in U.S. Pat. Nos. 5,741,650; 5,910,407; 6,149,529; 6,177,251; 6,203,993; 6,280,947; 6,300,077; 6,406,857; 6,440,661; 6,440,706; 6,448,002; 6,475,738; 6,482,595; 6,498,012; 6,503,718; 6,586,177; 6,919,174; 6,964,846; 7,811,757; 7,833,757; 7,915,015. Nevertheless, no single genetic change is known to be universally present in all colorectal cancers, therefore it became evident that only complex and expensive assays targeting a panel of multiple mutation markers in stool could detect CRC with the specificity approaching 95%. Nevertheless, the sensitivity of the mutation-based panel was only slightly above 50% (Imperiale et al., 2004).
Similarly, hypermethylation of regulatory sequences of several genes was suggested as an alternative or additional CRC biomarker (Jones et al., 2007; Wong et al., 2007). DNA hypermethylation detection in stool samples is described in recent U.S. Pat. Nos. 7,432,050; 7,485,420; 7,749,702; 7,785,772 as well as in WO2010/061023 and WO2010/089538. Although preliminary investigation of some methylation-related assays look promising (Glöckner et al, 2009; Hellebrekers et al., 2009; Nagasaka et al, 2009), DNA methylation assessment methods remain relatively complex and expensive.
Assessment of CRC-associated gene expression changes through analysing stool-derived RNA is emerging as another recent approach (Yu et al., 2008; Hamaya et al., 2010; Link et al., 2010). Methods of this type are already described in U.S. Pat. Nos. 6,258,541 and 7,816,077, however their clinical suitability remains questionable.
Despite growing interest in stool-based assays targeting nucleic acid-associated biomarkers of neoplasia, faecal material remains a problematic substance for detecting changes in the human DNA. The abundance of non-human, bacterial or food-derived DNA and the presence of substances interfering with PCR amplification are well-known problems related to stool sample analysis (Nechvatal et al., 2008). The importance of careful optimization of DNA isolation from stool samples needed to achieve reliable template amplification has also been repeatedly stressed (Whitney et al., 2004; Zou et al., 2007).
Likewise, several immunoassay-based techniques targeting biomarker proteins present in stool have been described in U.S. Pat. Nos. 5,380,647; 5,552,292; 5,695,945; 6,531,319; 6,703,206; 7,226,751; 7,252,955, 7,601,348. These approaches are not sufficiently developed to be used clinically for CRC diagnosis or screening.
Inflammatory bowel disease (IBD) is a group of common chronic disorders involving bowel inflammation. Ulcerative colitis and Crohn's disease are the most important conditions of this group. IBD is usually diagnosed in young adults. In most cases it is characterized by long remissions and incidental flare-ups usually requiring treatment. Currently the highest prevalence of IBD is observed in Europe (827 per 100,000 persons) and North America (568 per 100,000 persons) (Molodecky et al., 2012). All these patients should be monitored for a possible relapse. Those developing relapses are treated, and treatment efficiency assessment is an important task in need of serious improvements. In addition, there is a frequent necessity of distinguishing between extremely common functional gastrointestinal disorders, such as irritable bowel syndrome and IBD. Unfortunately, repeated diagnostic colonoscopies may be dangerous in IBD patients, and the range of non-invasive tests available for these purposes is very limited and includes only the use of serologic and faecal biomarkers, detection of which requires blood or stool collection and laboratory analysis (Foell et al., 2009). The most reliable method among those used for faecal sample analysis is the detection of neutrophil-specific protein calprotectin using ELISA assay described in U.S. Pat. No. 5,455,160 by Fagerhol et al.
It was also repeatedly demonstrated that in IBD patients inflammatory cells are present in abundance in the mucocellular layer overlaying colorectal mucosa (Loktionov, 2007: Loktionov et al., 2009; Loktionov et al., 2010; Anderson et al., 2011) and are excreted during defaecation.
The information presented above indicates that exfoliated cells, inflammatory cells and cell fragments found in the colonic mucocellular layer represent the informative element of the bowel contents in terms of detecting CRC and IBD, whereas the presence of stool-derived substances interferes with analytical procedures. For this reason attempts to obtain material less contaminated with faeces were undertaken by applying minimally invasive collection procedures involving a direct contact between a material-collecting surface and the rectal mucosa, which is the inner lining of the rectum.
Traditional digital rectal examination used in practical medicine for millennia is the simplest way of providing a contact between gloved finger of the examiner and patient's rectal mucosa. U.S. Pat. Nos. 4,857,457; 5,416,025; 6,187,591 proposed using this straightforward approach for obtaining rectal mucus, which is then analysed for the presence of a number of substances regarded as disease biomarkers.
In a more recent European Pat. No. EP1776048 a device equipped with an inflatable balloon, which is introduced to the rectum through a proctoscope is described. A rectal mucocellular layer sample is collected by inflating the balloon, which comes into contact with the surface of the rectal mucosa. Collection of well preserved colonic cells by this method was well documented in a few publications (Loktionov, 2007; Loktionov et al., 2009; Loktionov et al., 2010). The use of quantification of DNA isolated from collected material was successfully employed for CRC detection (Loktionov et al., 2009; Loktionov et al., 2010; Wallin et al., 2010), but this method is unlikely to be suitable for CRC screening purposes due to the proctoscopy requirement making the approach invasive.
Given the questionable basis of the FOBT, relying exclusively on blood presence detection (Itzkowitz, 2009), measurement of biomarkers obtained from cells found in the mucocellular layer of the large bowel appears to be the most promising analytical pathway to devising a really efficient non-invasive approach to CRC screening and detection of IBD. Unfortunately efforts of the scientific community in this direction were predominantly concentrated on stool-based methods with little thought devoted to the development of simple techniques for material self-collection by tested individuals and eventual creation of point-of-care tests or self-testing systems for CRC screening and IBD detection and monitoring.
Material self-collection and development of self-testing for CRC was considered in the context of FOBT by several authors. The generally accepted preparation of stool samples for FOBT normally involves temporary preservation of the whole excreted stool and transfer of its small portion on a special chemically treated card using a spatula, spoon or brush (see U.S. Pat. Nos. 3,252,762; 3,996,006; 4,092,120; 4,199,550; 4,333,734; 4,939,097; 4,562,043; 5,391,498; 5,563,071). A family of alternative FOBT modifications based on the use of faecal material obtained by wiping the anal area following defaecation with sheet-like soft multilayered composites was proposed by several authors. These versions of FOBT are described in the U.S. Pat. Nos. 4,259,964; 4,273,741; 4,367,750; 4,420,353; 4,559,949; 4,578,358; 4,578,359; 4,645,743; 4,808,379; 5,840,584; and European Pat. No. EP2108314. All these modifications providing sample self-collection were designed exclusively with the purpose of obtaining faecal material and detecting occult blood in it. Among them only the device described by Waldenburg in U.S. Pat. No. 5,840,584 could potentially be used for FOBT self-testing. Other early versions of FOBT designed for self-testing were based upon using indicator devices placed in the bowl of a toilet (U.S. Pat. Nos. 4,175,923 and 4,521,520). None of the latter devices has been used for clinical purposes as well as a point-of care version of the FOBT test on an examination glove proposed to be used immediately following rectal examination and described in U.S. Pat. No. 4,473,079.
The presented background information indicates that FOBT in different versions currently presents the only available option for non-invasive CRC screening. FOBT sensitivity and specificity are not sufficient, therefore an alternative test using more reliable CRC biomarkers than blood presence in stool could replace FOBT if shown to be more sensitive and specific than the latter, being at the same time inexpensive, simple and suitable for point-of-care or self-testing applications. Analysing the mucocellular layer containing exfoliated cells, cell fragments and biomolecules reflecting the state of the colorectal mucosa undoubtedly provides the best chance of detecting CRC presence. Likewise, IBD detection and inflammation activity assessment can be greatly facilitated by applying this approach. However, as discussed above, there are at present no methods for reliably sampling the mucocellular layer using non-invasive techniques. Therefore, the most challenging task in testing the mucocellular layer is to devise a non-invasive, simple and inexpensive technique for its sampling and analysis. The present invention addresses this problem.
For convenience, a list of references cited herein follows: